Method for mixing reagent and sample mounted on a slide

ABSTRACT

An automated immunostaining apparatus having a reagent application zone and a reagent supply zone. The apparatus has a carousel slide support (24) supporting a plurality of slide supports (26) thereon, and drive means (48) engaging the carousel slide support (24) for consecutively positioning each of a plurality of slide supports (26) in the reagent application zone. The apparatus also has a carousel reagent support (10) having a plurality of reagent container supports (11) thereon, and drive means (14) engaging the carousel for rotating the carousel and positioning a preselected reagent container support (11) in the reagent supply zone. The apparatus also has a reagent delivery actuator means (18) positioned for engaging a reagent container (12) positioned on a container support (11) in the reagent delivery zone and initiating reagent delivery from the reagent container (12) to a slide supported on a slide support (26) in the reagent receiving zone.

This application is a division of application Ser. No. 08/352,966, filedDec. 9, 1994, now U.S. Pat. No. 5,595,707; which is a continuation ofSer. No. 07/924,052, filed Aug. 31, 1992, now abandoned; which is acontinuation-in-part of Ser. No. 07/488,601, filed Mar. 2, 1990, nowabandoned.

TECHNICAL FIELD

This invention relates an improved biological reaction platform whichcan be used for a wide variety of assays, for example, automaticimmunostaining of tissue sections, in situ DNA analysis, immunoassayssuch as ELISA, and the like. The automatic device of this invention canbe used to process a large number of samples such as tissue sectionsmounted on slide surfaces using agents and protocols preselected by theoperator, while maintaining the slide surfaces in a substantiallyhorizontal plane throughout the incubation cycles.

BACKGROUND ART

Immunostaining and in situ DNA analysis are useful tools in histologicaldiagnosis and the study of tissue morphology. Immunostaining relies onthe specific binding affinity of antibodies with epitopes in tissuesamples, and the increasing availability of antibodies which bindspecifically with unique epitopes present only in certain types ofdiseased cellular tissue. Immunostaining requires a series of treatmentsteps conducted on a tissue section mounted on a glass slide tohighlight by selective staining certain morphological indicators ofdisease states. Typical steps include pretreatment of the tissue sectionto reduce non-specific binding, antibody treatment and incubation,enzyme labeled secondary antibody treatment and incubation, substratereaction with the enzyme to produce a fluorophore or chromophorehighlighting areas of the tissue section having epitopes binding withthe antibody, counterstaining, and the like. Each of these steps isseparated by multiple rinse steps to remove unreacted residual reagentfrom the prior step. Incubations are conducted at elevated temperatures,usually around 40° C., and the tissue must be continuously protectedfrom dehydration. In situ DNA analysis relies upon the specific bindingaffinity of probes with unique nucleotide sequences in cell or tissuesamples and similarly involves a series of process steps, with a varietyof reagents and process temperature requirements.

Automated systems have been explored to introduce cost savings,uniformity of slide preparation, and reduction of procedural humanerrors. Stross, W. et al, J. Clin. Pathol. 42:106-112 (1989) describes asystem comprising a series of baths positioned under the circumferenceof a circular, rotatable disc from which slide trays are suspended. Thedisc is lifted to lift slide trays from their baths, turned to positionthe slide trays above the next consecutive bath, and lowered to immersethe slide trays in the baths. This operation can be automated withsuitable timers and switches. This system exposes each of the slides tothe same treatment and relies on dipping for application of reactantsand rinsing.

Stark, E. et al, J. Immunol. Methods. 107:89-92 (1988) describes amicroprocessor controlled system including a revolving table or carouselsupporting radially positioned slides. A stepper motor rotates thetable, placing each slide under one of the stationary syringespositioned above the slides. A predetermined volume of liquid,determined by a dial, is delivered to a slide from each syringe.Microprocessor controls are provided.

Cosgrove, R. et. al., ACL. pp. 23-27 (December, 1989) describe animmunostaining apparatus for auto-pipetting reagents into a slide wellfrom a Carousel holding up to 18 reagent vials. Below each well, acoverplate spaced from the surface of each slide provides cover anddefines a reagent flow channel. The slides are suspended at a steepangle. Reagent from the well flows downward over the slide surface. Arow of slides are suspended for sequential treatment. Washing isaccomplished by a 3 to 4 minute continuous running wash over the sample,yielding an estimated 20:1 wash/reagent ratio.

Brigati, D. et al, J. Histotechnology 11:165-183 (1988) and Unger, E.,Brigati, D. et al, et al, J. Histotechnology. 11:253-258 (1988) describethe Fisher automated work station using capillary gap technology. Acoverplate is placed over the slide, forming a capillary gap. Liquid isintroduced into the capillary gap by placing the lower edge of theplate-slide pair in a liquid. Liquid is removed by placing the loweredge of the plate-slide pair on a blotter. The system is furtherdescribed in U.S. Pat. Nos. 4,777,020, 4,798,706 and 4,801,431. Thepreviously known devices are limited in theist performance and unable tosatisfy the needs for automated, high precision immunohistology.

It is an object of this invention to provide a device which providesmore rapid, reliable and more reproducible results than standardmethods; can perform any standard immunochemical assay including assaysrelying on immunofluorescence, indirect immunoassay procedures,peroxidase anti-peroxidase methods, or avidin-biotin technology;preforms all steps of the immunohistochemical assay irrespective ofcomplexity or their order, at the time and temperature, and in theenvironment needed; and is cost effective in terms of equipment, reagentand labor costs.

DISCLOSURE OF THE INVENTION

The automated biological processing apparatus of this inventioncomprises a reagent carousel cooperating with a sample support carouselto apply a sequence of preselected reagents to each of the samples withinterposed mixing, incubating, and rinsing steps cooperating therewith.The slide support carousel has a plurality of slide supports thereon anddrive means engaging the slide support carousel for consecutivelypositioning each of a plurality of slide supports in a reagent receivingzone. The reagent carousel has a plurality of reagent container supportsthereon and drive means engaging the reagent carousel for rotating thiscarousel and positioning a preselected reagent container support andassociated reagent container in a reagent supply zone. The apparatus hasa reagent delivery actuator means positioned for engaging a reagentcontainer positioned on a container support in the reagent supply zoneand initiating reagent delivery from the reagent container to a slidesupported on a slide support in the reagent receiving zone.

The apparatus preferably has bar code readers positioned to read barcodes on the sample containers or slides and on the reagent containers.Each of the carousels have homing systems containing a detectablecomponent and a proximity detector therefor for indexing the position ofthe reagent containers and slides.

One particular advantageous feature of the present invention is that byemploying a computer control arrangement to control the positioning ofthe reagent and slide support carousel, different reagent treatments canbe individually performed for each of the various tissue samples byappropriate programming of the apparatus. Additionally, the provision ofthe bar code readers permits tracking of each of the tissue samples aswell as a record of the reagents applied thereto.

The apparatus preferably has a heating chamber means surrounding theslide support carousel for heating slides supported thereon to apredetermined temperature. The heating chamber means includes a hot gasmanifold having a plurality of hot gas outlets positioned above theslide supports. The heating chamber means includes a temperature sensorand a hot gas control means connected to the temperature sensor forincreasing heat supplied to gas flowing through the manifold and forincreasing the hot gas flow rate if further heat is required to maintainthe heating chamber at a preselected temperature. The temperature sensoris a thermistor, the tip thereof being enclosed in a heat sensitivityreducing jacket. The hot gas control system includes two heatingcomponents with separate controls and a speed control for the hot gasfan.

The drive means engaging the slide support carousel is also a means forconsecutively positioning each of a plurality of slide supports at rinsezone, an evaporation control liquid and reagent receiving zone, a vortexmixing zone including vortex mixing means, and an incubation zone formedby the heating chamber means.

According to a first embodiment of the rinse zone, rinse spray means arepositioned adjacent to the rinse zone for applying pulses of rinseliquid to the surface of each of the slides positioned in the rinsezone. The apparatus slide supports are, according to this firstembodiment of the rinse zone, pivotally mounted for pivotal motion froma horizontal slide incubation position to a tilted slide drainingposition following each pulse of rinse liquid.

According, to a second embodiment of the rinse zone, first and secondrinse spray means are respectively positioned only at the beginning andend of the rinse zone, so as to be spaced from one another. The firstrinse spray means deposits a layer of rinse liquid onto a slide uponentering the rinse zone and the second spray means, after apredetermined waiting period, uses pulsed streams of rinse liquid,alternately directed at the longitudinal edges of the slides, to knockthe previously deposited layer of rinse liquid off of the slide as theslide exits the rinse zone. According to this second embodiment of therinse zone, the apparatus slide supports are stationary, a jet drainbeing provided at, for example, the end of the rinse zone, which directsa stream of fluid, such as, for example, air or the like, over the slideto drain any remaining rinse liquid off of the slide surface.

The apparatus preferably has a volumetric pump means, and a reagentdelivery actuator means positioned for activating the volumetric pumpmeans, thereby effecting delivery of reagent from a reagent container bythe volumetric pump to the reagent delivery zone. An evaporationinhibitor liquid application means is positioned adjacent the reagentdelivery zone.

Vortex agitation means are positioned adjacent the agitation zone forstirring reactants on a slide supported in the vortex agitation zone.

The pivoting slide support has distal and proximal ends, the distal endhaving raised terminal and lateral distal guide tabs with guide termini.The proximal end has first and second lateral guide tabs with opposedslide engaging surfaces for engaging and holding the lateral edges of aslide. The guide termini are lower than the upper slide surface plane.In this embodiment of the slide support, the slide support surface istipped or pivoted by a tipper to drain rinse liquid from the surface ofthe slide.

The stationary slide support has a slide support platform at a proximalend and a slide support post at a distal end thereof. The distal endalso has raised lateral distal guide tabs with guide termini betweenwhich a slide is positioned. The slide support platform at the proximalend has a guide edge and a slide clamping arrangement for clamping aslide to the support platform without interfering with the readingoperation of the bar code reader. The distal guide termini are lowerthan the upper slide surface plane to prevent wick-off of liquid on the,slide surface. In this embodiment, rinse liquid is drained from thesurface of the slide employing a jet drain which directs a stream offluid, i.e., gas or liquid, over the slide surface.

An improved biochemical method of this invention with increased sampledehydration protection comprises carrying out a biochemical reactionunder a, layer of evaporation inhibiting liquid. The improvementcomprises (a) covering the sample with an aqueous surface layer byapplying an aqueous solution to a planar support surface adjacent abiological sample mounted thereon; and (b) covering the aqueous surfacelayer with an evaporation inhibiting liquid layer by applying theevaporation inhibiting liquid to the planar support surface adjacent thebiological sample in an amount sufficient to form a continuous layer ofevaporation inhibiting liquid over the Sample. The evaporationinhibiting liquid is substantially water-insoluble, substantiallywater-immiscible and substantially non-viscous; has a specific gravityless than water, and a boiling point above 50° C.; and is devoid ofchemical characteristics which would significantly interfere withbiochemical reactions carried out on the sample. The biological samplecan then be optionally treated (c) with an aqueous reagent solution byapplying the reagent solution to the planar support surface adjacent thebiological sample. The reagent solution flows to the biological sampleunder the evaporation inhibiting liquid layer; and the sample iscontinuously protected from dehydration by the evaporation inhibitinglayer.

In another aspect of this invention, the reagent solution is stirred onthe surface of the biological sample by applying at least one gas streamto an area of the surface of the evaporation inhibiting liquid layerbetween the center of the evaporation inhibiting layer and the edge ofthe planar support surface, the gas stream having a central axis formingan acute angle with the planar support surface. According to oneembodiment of the present invention, the reagent solution is preferablestirred by a vortex formed by applying two off-center gas streams,flowing in opposite directions, to the surface of the evaporationinhibiting liquid layer. According to a further embodiment of thepresent invention, the reagent solution is stirred by a vortex formed byapplying a single gas stream along a longitudinal edge of the slide, thegas stream originating from the distal edge of the slide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a left front, isometric view of the automated immunostainingapparatus according to a first embodiment of this invention whichemploys a tipper rinse method, with the cabinet shell removed.

FIG. 2 is an exploded right front isometric view of the apparatus shownin FIG. 1.

FIG. 3 is a partial exploded left front, isometric view of the apparatusshown in FIG. 1.

FIG. 4 is a partial exploded right rear isometric view of the apparatusshown in FIG. 1.

FIG. 5 is a top view of a pivotally mounted slide support.

FIG. 6 is an isometric view of the underside of the slide supportcomponent.

FIG. 7 is a Side view of the pivotally mounted slide support of FIG. 5showing the tipper and mounting details.

FIG. 8 is an isometric view of the mounted slide support of FIG. 7 inthe untipped position.

FIG. 9 is an isometric view of the mounted slide support of FIG. 7 inthe tipped position.

FIG. 10 is a distal end view of the mounted slide support in the tippedposition.

FIG. 11 a fragmentary top view of the slide support carousel showingdetails of the slide treatment stations.

FIG. 12 is a schematic cross-sectional view of a rinse station takenalong the line A--A in FIG. 11, showing details of rinse liquid flow ona slide.

FIG. 13 is a top schematic view of the rinse stations showing details ofthe rinse liquid distribution on slides being treated therein.

FIG. 14 is an isometric view of the slide treatment bar code reading,rinse, reagent receiving and vortex mixing stations.

FIG. 15 is a schematic, fragmentary cross-sectional view of theevaporation inhibiting liquid and reagent receiving station, taken alongthe line B--B in FIG. 11.

FIG. 16 is a cross-sectional view of the vortex mixing assembly, takenalong the line C--C in FIG. 11.

FIG. 17 is a top schematic view of the vortex mixing zone, showingdetails of the vortex mixing action.

FIGS. 18A-C is a schematic representational cross-sectional view of aslide following the rinse liquid, evaporation inhibitor and reagentapplication steps.

FIGS. 19A-19B are cross-sectional views of respective alternativeembodiments of a rinse liquid container and associated heatingcomponents.

FIG. 20A is a bottom, isometric view of one embodiment of a reagentcontainer support tray.

FIGS. 20B-20C are side sectional views of a further embodiment of thereagent container support tray.

FIG. 21 is a fragmentary cross-sectional view taken along the line D--Din FIG. 11 showing the slide carousel metal proximity sensor indexingsystem of this invention.

FIG. 22 is a schematic view of the pneumatic system of the automatedimmunostaining apparatus of this invention.

FIG. 23 is a schematic drawing of the 120 volt AC power distribution inthe apparatus of this invention.

FIG. 24 is a schematic drawing of the DC power distribution in theapparatus of this invention.

FIG. 25 is a schematic drawing of a first portion of the computerdigital I/O system in the apparatus of this invention.

FIG. 26 is a schematic drawing of a second portion of the computerdigital I/O system in the apparatus of this invention.

FIG. 27 is schematic drawing of the computer serial and floppy disk I/Osystem in the apparatus of this invention.

FIG. 28 is a further embodiment of the intermediate section of theapparatus of this invention which dispenses with the tipper rinsemethod.

FIGS. 29A-29B are top and side views respective an alternativeembodiment of the slide support for use with the embodiment of FIG. 28.

FIG. 30A is a side, isometric view of one embodiment of a single washblock nozzle for use with the embodiment of FIG. 28.

FIG. 30B is a side, cross-sectional view of the single wash block nozzleof FIG. 30A.

FIG. 31 is a side, isometric view of one embodiment of a dual wash blocknozzle for use with the embodiment of FIG. 28.

FIG. 32 is a top view of a further embodiment of the vortex mixers foruse with the embodiment of FIG. 28.

FIGS. 33A-33B are side and front views respectively of bar code cleaningarrangement for use with the embodiment of FIG. 28.

FIG. 34 is a top, isomeric view of a jet drain.

BEST MODE FOR CARRYING OUT THE INVENTION

The automated immunostaining apparatus of this invention preforms allsteps of immunohistochemical and in situ DNA assays irrespective ofcomplexity or their order, at the time and temperature, and in theenvironment needed. Specially prepared slides containing a bar codeidentifier and a mounted tissue section are placed in special support ona carousel, subjected to a preprogrammed sequence of reactions, and areremoved from the carousel, ready for coverslipping and histologicalexamination. For purposes of clarity of the following description of theapparatus of this invention and not by way of limitation, the apparatuswill be described in terms of immunohistochemical processes.

FIG. 1 is a front right, isometric view of the automated immunostainingapparatus of this invention, with the cabinet shell removed. Liquid andair supply tubing and electrical wiring connecting the respectivecomponents are conventional, well known in the art, and are omitted fromthe drawings for purposes of clarity. The apparatus has an upper section2, intermediate section 4 and lower section 6. In the upper section 2,reagent bottle support carousel 10 is mounted for rotation about itscentral axis 7 on upper support plate 8. Reagent bottles 12 required forthe immuno-histochemical reactions to be conducted during slidetreatment cycle are supported by the carousel 10, mounted in reagentbottle receptors 11. These receptors 11 are configured to receivevolumetric pump outlet tube 307, shown in detail in FIG. 15. Thereceptors 11 are preferably equally spaced in a circular pattern axiallyconcentric with the carousel axis 7. The number of receptors 11 providedshould be sufficient to accommodate the number of different reagentbottles 12 required for a cycle or series of cycles. Twenty-fivereceptors 11 are shown, but the number can be smaller or greater, andthe diameter of the carousel 10 can be increased to accept a largernumber of reagent bottles 12. The carousel 10 is rotated by the steppermotor 14 drive belt 16 to a position placing a selected reagent bottle12, in the reagent deliver position under the air cylinder reagentdelivery actuator 18 over a slide to be treated with reagent. Reagenttray motor driver 20 is connected to stepper motor 14.

The intermediate Section. 4 comprises support plate 22 upon which theslide support carousel 24 is rotatably mounted. The carousel 24 supportsslide supports 26. Heated air supply chamber 28 communicates with theheated air supply manifold 30 supported on the underside of plate 8 andlid heated air supply manifold 31 mounted on the upper plate 8 by hingedsupports 33. The support plate 22 also supports the conventionalcomputer board 32, LCD display 34, disk drive 35 and computer 36 used tooperate the apparatus. Air pressure regulator 38, as best seen in FIG.2, regulates the pressure of air delivered to the evaporation inhibitorand rinse liquid delivery systems described in FIG. 22.

The lower section 6 includes support plate 40 upon which are supportedaccessories such as power supply filter 42 and hot water supply 44.

FIG. 2, FIG. 3 and FIG. 4 are exploded right front, left front and rightrear isometric views of the apparatus shown in FIG. 1. Tipper aircylinders 46 are positioned on support plate 8. These cylinders arealigned to actuate a tipper cam surface 148 against a elide support tabsurface 112 shown in detail in FIGS. 8, 9 and 10.

In the intermediate Section 4, the stepper motor 48 rotates the slidesupport carousel 24, engaging drive belt 25 (FIGS. 3 and 4) engaging theperimeter of the slide support carousel 24. Splash guard 50 is a wallwhich surrounds the sides, back and part of the front of the carousel24, defines the heating zone and contains the liquid spray and dropletsproduced in the processing. It extends upward from the intermediateplate 22 to a position adjacent the upper plate 8, leaving an air flowgap between the upper edge of the splash guard 50 and the underside ofthe plate 8. Mounted on the underside of upper support plate 8 above thecarousel 24 and within the perimeter of the splash guard 50 is theheated gas supply manifold 30 (FIG. 2). Heated air is directed downwardand over the slide supports 26 by holes 336 (FIG. 15) in the manifold30. The heated air then passes upward over the top of the splash guard50 and exits the device. Extending upward through central opening 52 ofcarousel 24 into the heated air supply chamber 28 is the fan shroud 54and axially positioned fan 56. The fan 56 is positioned over air vents57 in the bottom plate 22. The annular waste liquid sump 58 surroundsthe shroud 54, below liquid outlet ports 292 (FIG. 14), and is supportedon the bottom of plate 22. The waste reagent and rinse liquids arecollected in the sump and passed to a drain through an outlet tube, inthe sump bottom (not shown).

Rinse and liquid coverslip spray blocks 60 are supplied with liquidthrough conventional solenoid valves 62.

Temperature controller 66, mounted on support plate 22, controls theheat energy supplied to the heated water container 44. Temperaturecontrollers 68 and 70 mounted on support plate 40 (FIG. 4), control thetemperature of the air in the heated air supply chamber 28 bycontrolling energy supplied to respective annular heater elements 331and 332 (FIG. 15). Slide carousel stepper motor driver 72 and relay 74operate stepper motor 48. Power supplies 76 and 78 provide power to thestepper motors and control systems. Air compressor 80 supplies air tothe air filter 82 and air pressure regulators 38, 64 and 86.

FIG. 5 is a top view of a first embodiment of a mounted slide support 26with slide edges 100 and 101 represented by dashed lines. The slidesupport 26 has a support plate 102 with a distal end 103 and a proximalend 104. The distal end 103 has a raised terminal guide end tab 106 andtwo lateral guide tabs 108 and 110 with the upper edges constitutingguide tab termini. The distance between the upper surface of the slidesupport. 26 and the guide tab termini (the elevation above the uppersurface) is less then the thickness of a conventional microscope slide.The proximal end 104 of the slide support 26 has opposed lateral guides112 and 114 for engaging the lateral edges of a slide and a terminal endtab 115 for engaging the proximal end of a slide. The proximal end 104of the slide support 26 has an inflexible support portion 116 providinga lateral edge 120 and a flexible arm 118 including a lateral edge 122positioned such that lateral edges 120 and 122 oppose one another. Thedistance between the slide edge engaging surfaces 111 and 113 of theguide tabs 112 and 114 is less than the width of a slide to be supportedon the slide support 26. A standard slide has a width of 1 inch or 25mm, and the preferred distance between the slide edge engaging surfaces111, 113 of the tabs 112, 114 for supporting a standard slide is from 20to 24 mm. The flexure of arm 118 permits positioning of the slidebetween the lateral guide tabs and terminal end tabs 106, 115. Thedistance between the opposing tab surfaces 111 and 113 causes the slidesupport 26 to apply a positive pressure on the edges of a slide,retaining the slide securely on the slide support 26 during the tiltingand other processing steps. The upper surface of the support plate 102is preferably planar and smooth so the wet slide rests closely on thesurface 102, and surface tension will resist vertical movement of theslide from the support Surface 102.

FIG. 6 is an isometric view of the underside of the slide support 26.The inflexible portion 116 has an integral pivot support 124 whichreinforces the inflexible portion 116 to prevent flexure. The flexiblearm 118 has sufficient depth or thickness to limit the flexural movementof the arm 118 to a horizontal direction. This insures effectivecooperation and pressure between the guide tab 112 on the inflexibleportion 116 and the guide tab 114 on the flexible arm 118 to assist inretaining the slide in place on the slide support 26 during the tippingoperation described in detail hereinafter.

FIG. 7 is a side view of a mounted slide support showing the tipper andmounting details. The upper pivot support 124 is pivotally mounted onthe lower pivot support 126. Lower pivot support 126 has upwardextending projections 128 and 130 which engage the ends 132 and 134 ofthe upper pivot support 124. Pivot pin 136 extends through an axiallyaligned hole in projection 128 into an axially aligned receptor hole 138(FIG. 6) in the opposing end 132 of the upper pivot support 124. At theopposite end, axially concentric with pivot pin 136, pivot pin 140extends through a hole in projection 128 (not shown) into a respectivereceptor hole in the opposing end 134 of the upper pivot support 124.The slide support 102 is thus mounted for pivotal motion around thecommon pivot axis of the pins 136 and 140. Bias spring 142 is supportedon pin 134, one end 141 pressing against the lower abutment surface 143of the inflexible support portion 116, and the other end 144 bearingagainst spring stop groove 145 in the spring stop 146. The tip 148 oftipper 150 is positioned above the upper surface of guide tab 112 whenthe slides are positioned in a rinse station, described in greaterdetail hereinafter with respect to FIG. 13.

The pivot pins 136 and 140 support the Upper surface of the slidesupport 102 at a small angle `a` from the horizontal plane to aid liquidflow toward the distal end 103 during treatment. Angle `a` is preferablyin the range of from 0.3° to 1.0°. The upper surface 151 of theinflexible support portion 116 and the upper slide surface 152 (dottedline) supported thereon are thus maintained at a slight incline from thehorizontal plane downward toward the distal end 103 of the Slide support26.

FIG. 8 is an isometric view of a slide (dashed lines) mounted on slidesupport 26 in the untipped position, FIG. 9 is an isometric view of themounted slide support 26 in the tipped position, and. FIG. 10 is adistal end view of the mounted slide support 26 in the tipped position.Vertically downward pressure of the tipper tip 148 against the upperguide tab surface 154 of guide tab 112 rotates the support plate 102about the pivot axis 156 defined by the pivot pins 136 and 140. Thepivot axis 156 (FIG. 5) preferably lies in a vertical plane through themidpoint of distal end 103 and the left edge proximal end 104 of theslide support 26. The tipping action tilts the slide surface to an angle`c` of approximately 60° from the vertical (FIG. 10). It sharply lowersdistal corner 158 and sharply raises proximal corner 160, breaking theliquid meniscus on the slide surface and directing the liquid flow 159to the corner 158 and off the surface of the slide into drain hole, 292.The pivotal movement increases the pressure of the spring 142 againstspring stop groove 145, and as the tipper 150 is raised, the slidesupport 25 returns to its Original position. The slide support returnpivot motion is terminated when distal corner 162 of the support plate102 abuts stop surface 164 of the lower pivot support 126.

FIG. 11 a fragmentary top view of the slide support carousel 24 showingdetails of the various slide treatment stations. Rinse nozzle blocks200, 202 and 204 and the adjacent respective slides 206, 208 and 210define successive rinse zones, details of which are shown in FIGS.12-14. Evaporation inhibitor liquid application block 212 and theadjacent slide 214 define the evaporation inhibitor and reagentapplication zone, details of which are shown in FIG. 15. Air cylinderreagent delivery actuator 18, supported by support arm 216, contactsreagent bottle 218, directly over slide 214. Vortex mixer air jet blocks220, 222 and 224 are positioned adjacent slides 226 and 228 in theagitation zone, details of which are shown in FIG. 16 and 17. The hanger352 is mounted on the tip of blocks 220 and 222 and supports suspendedblock 224. Pressurized air is delivered to block 224 by conduit 358. Asthe slide support carousel 24 positions each slide for successivetreatment in the rinse zones, evaporation inhibitor and reagentapplication zone, and agitation zones (counterclockwise movement of thecarousel), the tissue sections on each slide are first rinsed and thencovered with evaporation inhibitor. Reagent is applied from apreselected reagent bottle to the tissue through the evaporationinhibitor layer, and the reagent is agitated through the evaporatorinhibitor layer by the vortex mixer. Each slide then is moved around theincubation zone, a circular path traveled by the slide support carousel24, heated with hot air from the heated air manifold 30, and the reagentreacts with the sample. As the carousel 24 continues to increment aroundthe circle, each slide is returned to the rinse stations, etc, forapplication of the next reagent required in the reaction. This entirelyautomated process continues Until the desired reactions are completed.

Bar code reader 231 (FIG. 14) above slide 205 reads a slide bar code 233(FIGS. 13 and 17) on each slide. The slide bar codes 233 identifies theslide sample and the particular immunohistochemical process required forthat sample. This information is fed into the computer and correlatedwith the indexed position of that slide with respect to "home", tocontrol the sequence of reagents chemicals to be applied to that slidein the reagent application zone.

FIG. 12 is a schematic cross-sectional view of a rinse station takenalong the line A--A in FIG. 10, showing details of rinse liquid flow ona slide. Rinse block 200 mounted on plate 22 has a heated rinse liquidsupply channel 230 communicating With rinse liquid nozzle 232. The slide234 has a sloping surface at an angle `a`, being supported on thesloping surface of the slide support 102. The slide 234 has a rinseliquid impact zone 236 adjacent the proximal end 104 between the barcode 233 and the sample 238. The impact zone 236 is at a higherelevation than the tissue section 238 supported adjacent to distal end103. The nozzle axis 240 has an angle `b` which directs liquid againstthe slide surface impact zone 236. The impact zone 236 is above thetissue section 238 on the sloped surface of slide 240, and the rinseliquid stream 242 flows across the upper surface of the tissue section238 toward the distal end 103. The angle `b` preferably has an angle offrom 15° to 35°, and the distance between the exit of nozzle 232 and theslide 124 is selected to direct the rinse liquid precisely on the impactzone 236, avoiding disturbance of the fragile tissue section 238.

The slide support carousel 24 is rotated above the plate 22, the outerperiphery being supported by low friction slide bearings 244 arrayed inan axially concentric circular path on plate 22 under the outerperiphery of carousel 24.

FIG. 13 is a top schematic view of one embodiment of the rinse stationsshowing details of the rinse liquid distribution on slides being rinsedtherein. Slider 234, 246, and 248 are positioned in the path of heatedrinse solutions (dotted lines) from rinse station blocks 200, 202 and204. Fragile tissue sections 238, 250 and 252 are positioned adjacentthe distal end of the slides. The rinse liquid impact zones 236, 254 and256 are positioned between the tissue sections and the proximal ends ofthe slides, to avoid direct impact of the liquid jets from the rinseblock nozzles. The rinse nozzles on each block are preferably 11.5 mmapart. Rinse block 200 has right offset nozzles 232 and 258 (offset 2 mmto the right of center) supplied by channel 230 connected to supplytubing 260. This directs the rinse fluid toward the right surface of theslide, effecting a transverse flow path across the tissue section 238 tothe distal end drain corner 158. Rinse block 202 has symmetrical nozzles262 and 264 supplied by channel 266 connected to supply tubing 268. Thesymmetrical nozzle configuration effects a central flow path across thetissue section 250. Rinse block 204 has left offset nozzles 270 and 272(offset 2 mm to the left of center) supplied by channel 274 connected tosupply tubing 276. The left offset nozzles 270 and 272 direct a rinseflow path down the left side of the tissue section 252. The nozzlepatterns provide effective rinse solution flow distribution across allportions of the tissue section surface as the slide is treated in eachsuccessive rinse section.

FIG. 14 is an isometric view of the rinse stations, a evaporationinhibiting liquid and reagent application station, and agitationstations, showing details of the slide tipping action in the rinsesections. Tipper air cylinders 46 (FIG. 3 and 4) comprises threeconventional air cylinders 278, 280 and 282 with internal pressurizedair activated pistons or equivalent actuators. Pressurized air deliveryto the cylinders causes respective tipper tips 148, 284 and 286 to movedownward, pressing against respective slide support tabs 112, 288 and290. Three tipper positions are shown to illustrate the action thereof.Tipper tip 148 is shown in the fully withdrawn or resting position, andslide 206 is in the rinse solution receiving position. After applicationof heated rinse solution, the tipper descends through an intermediateposition shown by tipper tip 284 and slide support 208, to the drainposition shown by tipper tip 286 and slide support 210. Liquid drainsfrom the left distal corner (lowest corner) into a drain hole 292.

In each rinse station, the sample is treated with a repeated, preferablyat least seven, rinse cycles. Each rinse cycle comprises application ofapproximately 500 μL of heated rinse solution in a short pulse (120msec) to the slide, followed by tipping the slide to drain away therinse solution. An estimated 150 μL of liquid remains on the slide afterdraining. These rinse cycles are repeated in each rinse station. Theshort rinse pulse followed by draining prevents the formation of aequilibrium solute boundary layer and greatly increases the rinseefficiency, overcoming the boundary problems present in the prior artrinse methods. Assuming that 150 μL of rinse solution is left after eachdraining step, a 23 percent dilution is achieved with each rinse cycle.Thus the effective dilution in the combination of the three rinsestations is estimated to be 0.2 parts per trillion, many orders ofmagnitude more effective than prior art, biochemical rinse procedures.This greatly increases the sensitivity of the immunohistologicalprocess.

FIG. 15 is a schematic, fragmentary cross-sectional view of theevaporation inhibiting liquid and reagent application station, takenalong the line B--B in FIG. 11. Evaporation inhibitor liquid distributorblock 212 has a supply channel 293 and outlet nozzles 294.

The evaporation inhibiting liquid is substantially water-insoluble,substantially water-immiscible and substantially thin or non-viscous. Ithas a specific gravity less than water, and a boiling point above theprocess temperature, preferably above 100° C. It should be devoid ofchemical characteristics which would significantly interfere withbiochemical reactions carried out on the sample, that is, the reactionstaking place between the reagents and tissue sample on the slide.Preferred evaporation inhibiting liquids are hydrocarbons, optimallynon-aromatic saturated hydrocarbons, having from 9 to 18 carbons, mostoptimally having about 10 to 14 carbon atoms.

A small quantity of evaporation inhibitor liquid is directed by nozzle294 in a inhibitor liquid stream 296 to an impact zone 298 on the slidebetween the tissue sample 238 and the proximal end. 100 of the slide, sothat the tissue sample is not disturbed. The evaporation inhibitorliquid flows across the surface of the water layer on the wetted tissue,forming a thin evaporation inhibiting film 299 over the aqueous layerwhich usually covers most of the upper surface of the slide. The tissueis now ready for application of reagent.

The reagent delivery combination includes a conventional air cylinder 18or equivalent actuator having an internal pressurized air activatedpiston. It is supplied with pressurized air by tubing 300. Air cylinder18 is supported by plate 216 and post 302 mounted on upper plate 8.Delivery of pressurized air to the cylinder 18 causes rod 304 and itsattached foot 306 to move downward against a reagent container 12positioned in the reagent delivery zone. Downward movement of reagentcontainer 12 causes emission of a precise volume of reagent liquid 310.Suitable volumetric pumps are available from S. A. Valois and aredescribed in U.S. Pat. No. 4,245,967 and French patent 2,528,122.

The reagent carousel support 314 is the drive plate which supports thereagent bottle carousel 10 and rotates it about its axis to place apredetermined reagent bottle 12 in the reagent delivery zone. An axiallyconcentric circular array of low friction slide bearings 316, mounted onthe upper plate 8, are positioned under the outer edge of the reagentsupport carousel.

The predetermined volume of aqueous reagent 310 impacts the evaporationinhibitor surface film between the impact zone 298 and the upper edge ofthe tissue sample 299, passing through the film to the aqueous layerbeneath the film and reaching the slide surface. The reagent then flowsacross the tissue sample 238 under the covering film of evaporationinhibiting liquid 299. In this sequence, immediately after leaving therinse stations, the slide covered with the protective film to preventany dehydration of the tissue sample 299. The reagent solution is thenapplied to the protected tissue. Dehydration of the tissue section wouldirreversibly alter its physical and chemical characteristics and impairthe immunohistochemical reactions. Dehydration is a constant hazardbecause of the constant flow of heated air over the slides required tomaintain them at the desired temperature. The heated air temperature isdetermined by the requirements of the biochemical processes required bythe process. It is slightly above 40° C., preferably about 45° C., forimmunochemical reactions and can be as high as from 93° to 97° C. for insitu DNA hybridization reactions.

FIG. 15 also shows detailed elements of the heated air supply chamber 28shown in FIG. 1. Air is moved upward into the central intake manifoldchamber 330 and through annular heating coils 331 and 332 mounted onannular air passageway plate 333, to heat the air to a temperatureslightly above 40° C., preferably about 45° C. A higher temperature canbe provided as needed for in situ DNA hybridization procedures. Theheated air passes through the outlet manifold chamber 334 and out theoutlet passageways 336 in the lower plate 338. Annular, axiallyconcentric inner and outer heated air flow control curtains 340 and 342direct the heated air downward over the slide surface. The reagent 310falls through manifold passageway 344 to the slide surface.

The air temperature is monitored by heat sensor 345 positioned in thepath of the heated air. A preferred heat sensor is a thermistor encasedin a heat sensitivity adjusting jacket 347 which reduces the sensitivityof the thermocouple and approximates the thermal mass of the slides.

A reagent bar code reader 346 can be mounted on post 302, positioned toscan a reagent bar code 348 on the reagent bottle 12. Bar code 348identifies the contents of the reagent bottle. At the beginning of aslide treatment operation, the reagent carousel 10 is rotated past thebar code reader 346, and the bar code 348 on each reagent bottle 12 isscanned. The scanned information is fed to the computer and correlatedwith the indexed position of the reagent carousel 10. This informationis used to rotate the reagent carousel 10 to place the correct reagentbottle 12 in the application zone for each slide treatment step for eachslide.

FIG. 16 is a cross-sectional view of one embodiment of the vortex mixingassembly, taken along the line C--C in FIG. 11. Outer vortex jet block222, mounted on plate 22, has an pressurized air supply channel 350 andnozzle 351. Nozzle hanger 352 is mounted on the top of vortex block 22and supports suspended inner vortex air jet nozzle block 224. Channel354 supplies nozzle 355 in block 224 with pressurized air. Nozzles 351and 355 have central axes which form angles `d` and `e` of from 5° to15° with the horizontal, directing air jets 356 and 357 toward the slidesurface at the corresponding acute angles;

FIG. 17 is a top schematic view of the vortex mixing zone, showingdetails of the vortex mixing action. Pressurized air is supplied to thenozzle channels 350 and 354 by channel 358. The reagent solution coveredby a layer 360 of evaporation inhibiting liquid 360 is stirred on thesurface of the biological sample by applying at least one gas stream 356or 357 to an area of the surface of the evaporation inhibiting liquidlayer 360 between the center of the evaporation inhibiting layer 360 andthe edge of the planar support surface 361 or 362 of the slide 228. Thegas stream impacts the surface of the evaporation liquid surface layer360 and moves the underlying reagent solution in a circular path on thetissue section. Preferably, the reagent solution is stirred on thesurface of the biological sample by a vortex formed by applying two gasstreams 356 and 347. Stream 356 is directed against a area 363 of thesurface of the evaporation inhibiting liquid layer between the center ofthe evaporation inhibiting layer and the slide edge 361. Stream 357, ina direction opposite to the direction of stream 356, is directed againstan area 364 of the surface of the evaporation inhibiting liquid layerbetween the center of the evaporation inhibiting layer and the slideedge 362. Although this method is shown with respect to an evaporationliquid inhibitor covered reagent layer, it will be readily evident thatit can be applied to gently stir any liquid layer overlying a fragilesubstance.

FIG. 18 is a schematic representational cross-sectional view of a slide370 following the rinse liquid, evaporation inhibitor and reagentapplication steps. Following the rinse stages (Stage A), the tissuesection 371 mounted on slide 370 is cowered with a thin residual aqueouslayer 372. Following application of the evaporation inhibitor liquid(Stage B), the aqueous layer 372 and tissue section 371 is entirelycovered by a layer 373 of the evaporation inhibitor liquid. Aqueousreagent 374, applied to the slide, flows under the evaporation inhibitorlayer 373 to cover the tissue section. In the vortex mixing section(Stage C), air jets directed against the surface of the evaporationinhibitor liquid 373 move it and the reagent solution 374 thereunder ina swirling or stirring action on the surface of the fragile tissuesection. This gentle stirring achieves increased interaction of reagentwith the tissue section while preserving the tissue from dehydration orother damage from the air jets.

FIG. 19A is a cross-sectional view of one embodiment of a rinse liquidcontainer and associated heating components. The rinse liquid applied tothe surface of the slides by rinse blocks 200, 202 and 204 should have atemperature above 40° C. and is preferably about 45° C. The elevatedtemperature is critical for the immunochemical reactions. The rinseliquid is supplied by the hot water supply 44. The hot water supply 44comprises an inner container of an inert material having a lowcoefficient of expansion such as a pyrex bottle 382 having a threadedneck 384 to which a cap 386 is attached by threads. The container 382 issurrounded by an insulating jacket 388 of suitable insulation materialsuch as a fiberglass layer. Between the insulating jacket 388 and thebottle 382 is a heating jacket 390 with electrical power leads 392. Asuitable heating jacket is a thick sheet of silastic rubber(polysiloxane) with embedded resistance heating coils having a combinedheating value of about 180 watts. A conventional safety thermostat 394,connected to the elements of the heating jacket, is also providedbetween the insulating jacket 388 and bottle 382. The safety thermostatprevents the rinse liquid temperature from exceeding a preset value,preferably about 50° C. A thermistor temperature sensor 391 with leads393 extends through the cap 386 into the upper zone of the bottle 382.An liquid inlet tube 394 extends through the cap 386 to the bottom ofthe neck 384, and an outlet tube 396 extends through the cap 386 to thebottom of the bottle 382.

This unique configuration provides a highly uniform liquid outputtemperature. The colder water entering through the inlet tube 394, beingmore dense than the heated liquid in the bottle, sinks downward past theheated container walls and is heated. The displaced liquid rises upwardin the container. This stirring motion thoroughly mixes the liquidwithout the need for an agitator, producing a highly uniform outletliquid temperature. Thermistor 391 constantly monitors the liquidtemperature, providing a signal to the control system which is used todetermine when the heating elements in jacket 390 should be energized.

FIG. 19B illustrates an alternative embodiment of the rinse liquidcontainer and associated heating components of the present which issimilar to the structure illustrated by FIG. 19A except that the inlettube 394 of the embodiment of FIG. 19 functions as an outlet tube 394Aand outlet tube 396 of the embodiment of FIG. 19 functions as an inlettube 396A, i.e., the inlet and outlet lines have been reversed. Thisarrangement prevents the build up of air or gas in the bottle 384.Additionally, the inlet tube 396A has been provided with perforations396B for obtaining mixing as the bottle 384 is replenished with liquid.

FIG. 20A is a bottom, isometric view of one embodiment of a reagentcontainer support carousel 10. According to this embodiment, the reagentcontainer carousel 10 has feet 800, 801 and 802 which rest in respectivematching recesses in the reagent carousel support 314 (FIG. 15) in onlyone position. This insures that the reagent carousel 10A and the reagentbottle receptors 11 are always positioned in predetermined orientationon the carousel support 314.

The feet 800, 801 and 802 also function as supporting feet when thereagent support carousel 10 is removed. Refrigeration of the reagents isoften required during their storage. The reagent container carousel 10,with the reagent bottles supported thereon, can be lifted from thecarousel support 314 and placed in a refrigerator, supported by the feet800, 801 and 802.

Indexing metal homing block 803 is mounted on the reagent containercarousel 10 and rotates with the carousel 10. A conventional metalproximity detector (not shown) is mounted on the upper plate 8 at anposition which places it adjacent the rotational path of the homingblock. A change in electrical signal from the proximity detectorindicates that the metal homing block is in the `home` position adjacentthe block.

FIG. 20B is an alternative embodiment of a reagent support carousel 10Aand associated carousel support 314A wherein a handle 804 has beenprovided to assist in the removal and replacement of the reagent supportcarousel 10A as described above. In this embodiment, the carousel 10A isprovided with a plurality of feet 800A, for example, five feet, Whichare substantially cylindrical elements with beveled edges 805, and fitinto corresponding and matching circular openings 802A, formed in theassociated carousel support 314A. The feet 800A and opening 802A arepositioned so that the carousel 10A will fit into the support 314A inonly one position such that the carousel 10A is always positioned in apredetermined orientation on the support 314A. The support 314A isprovided with a central hub 806 which is received in a central opening807 formed in the carousel 10A, the hub being provided with bevelededges 808. Engagement of the carousel 10A and the support 314A is bestseen in FIG. 20C. Except for the above described differences, thecarousel 10A and the support 314A are the same as previously described.

FIG. 21 is a fragmentary cross-sectional view taken along the line D--Din FIG. 11. Indexing block 229 is a metal block. Proximity, sensor 610is supported on the underside of plate 22 by bracket 611. The proximitysensor 610 emits an electrical signal through leads 612 which changeswhen the metal block 229 is positioned in the `home` positionimmediately above the sensor.

The homing systems of the reagent carousel 10 and slide support carousel24 operate in a similar manner. Presence of an indexing block adjacentthe sensor produces a signal indicating that the carousel is in a "home"position, and provides a reference for subsequent indexed movements ofthe respective stepper motor drive and subsequent indexed movements ofthe respective carousel.

FIG. 22 is a schematic view of the pneumatic system of the automatedimmunostaining apparatus of this invention. The air supply for thesystem is supplied by air compressor 80 and air filter 82. The outputline 400 from the air filter 82 is connected to the input port of airpressure regulator 86 where it is regulated to a constant outputpressure of about 25 psi. Diaphragm pressure switch 402 ommunicates withthe air pressure regulator 86 outlet line 403 through line 404.Diaphragm pressure switch 402 closes the system circuit breaker 406 whenthe pressure in line 404 is at least 22 psi. Failure of the aircompressor and resulting drop in line pressure automatically deactivatesthe system.

The air output branch line 408 lead is connected by line 410 with tipperair cylinder three way control solenoid valve 412. When in an "open"position, solenoid valve 412 provides communication between input lineand cylinder 278. This permits pressurized air to pass from line 410 toair cylinder 278, thus pressing tipper tip 148 (FIG. 14) against therespective slide support tab 112 and tipping the slide support 206. Whensolenoid valve 412 returns to the vent position, the air cylinder 278communicates with atmosphere, permitting the air cylinder 278 to returnto its resting position. Tipper tip 148 then rises to its restingposition, allowing the slide support to also return to its horizontalposition. Three way solenoid valves 416 and 420 operate in an identicalway, providing communication between the air inlet lines 414 and 418 andthe respective air cylinders 280 and 282 when in the open position andactuating respective tipper tips 284 and 286. They also opencommunication between the air cylinders 280 and 282 and the atmospherein the vent position, allowing the tipper tips to return to theirelevated position.

Branch line 422 leads from line 408 to the reagent dispenser three waycontrol solenoid valve 424. When energized to an "open" position,solenoid valve 424 permits pressurized air to pass from line 422 to aircylinder input line 300, causing rod 302 and foot 306 (FIG. 15) to pressthe reagent dispenser bottle 12 downward, emitting a precise volume ofreagent liquid. When solenoid valve 424 is in the vent position, the aircylinder 18 and the reagent bottle 12 return to their resting positions.

Branch line 426 leads from line 403 to branched lines 428 and 430.Branch line 428 leads to pressure regulator 38, providing an outputpressure of 10 psi in output line 431. Three way solenoid valve 432,when in the open position, provides communication between air input line431 to the evaporation inhibitor liquid reservoir container 434 throughlines 436 and 438. It also delivers pressurized air to the rinse liquidsupply container 44 through line 440, rinse solution reservoir 441 andsupply conduit 443. When solenoid valve is opened to atmosphere (ventposition), air in line 436 and in containers 44 and 434 is bled orvented to the atmosphere. This permits removal, opening or replacementof reservoir container 434, or opening or removal of supply container441. The pressured air in containers 434 and 441 forces liquid throughrespective output conduits 442 and 443.

Conduit 442 leads to two way solenoid valve 446, which has an outletconduit 448 leading to the evaporation inhibitor application block 212and associated nozzles. When the solenoid 446 is opened evaporationinhibitor liquid is emitted from nozzles 294 (FIG. 14 and 15) onto thesurface of the respective slide 234.

Conduit 444 delivers pressurized rinse liquid from heated rinse liquidcontainer 44 to branch conduits 450, 452 and 454 leading to conventionalrinse liquid two way solenoid valves 460, 462 and 464. When the solenoidvalves 460, 462 and 464 are opened, pressurized rinse liquid isdelivered to the respective rinse blocks 200, 202 and 204 through supplyconduits 260, 268 and 276. The pressurized rinse liquid is emitted bythe rinse blocks onto the slides positioned in the respective station(FIG. 13).

Branch line 430 leads to pressure regulator 64, providing an outputpressure of 15 psi in output conduit 466 leading to vortex mixer aircontrol two way solenoid valve 468. When in the open position solenoidvalve 468 delivers pressurized air to output conduit 470 connectedthereto. Conduit 470 leads to branch lines 472 and 474 leading to vortexmixing blocks 222 and 224. The pressurized air is emitted by nozzles 351and 355 (FIG. 17), stirring the reagent layer on the respective slides234.

FIG. 23 is a schematic drawing of the 120 volt AC power distribution inthe apparatus of this invention. The power circuit to power line filter500 includes a main fuse 504 and main power switch 506. 120 Volt ACpower to the air compressor 80 is provided by line 511 from the linefuse 510 in the I/O board 508. 120 Volt AC power to the air compressorcooling fan 514 is provided by line 513 from line fuse 512 in the I/Oboard 508. 120 Volt AC power to the electronics cooling fan 518 isprovided by line 517 from line fuse 516 in the I/O board 508. 120 VoltAC power to the 24 volt DC power supply is provided by line 521 fromline fuse 520 in the I/O board 508. 120 Volt AC power to the 5 volt/12volt DC power supply 78 is provided by line 524 from line fuse 522 inthe I/O board 508. 120 Volt AC power to the computer card rack 529 isprovided by line 528 from line fuse 526 in the I/O board 508. 120 VoltAC power to slide heater fan relay 533 is provided by line 532 from linefuse 530 in the I/O board 508. 120 Volt AC power to the slide heaterrelays 537 is provided by line 536 from fuse 534 in the I/O board 508.120 Volt AC power to the rinse fluid heater relay 541 is provided byline 540 from fuse 538.

FIG. 24 is a schematic drawing of the DC power distribution in theapparatus of this invention. 12 Volt DC logic power for printer 550 isprovided by line 552 from the power supply 78. Similarly, 12 volt DCpower for low slide temperature controller 68 is provided by line 554,12 volt power for high slide temperature controller 70 is provided byline 556, and 12 volt power for rinse fluid temperature controller 66 isprovided By line 558. 5 Volt DC laser power for the slide bar codereader 231 is provided by line 560 from the power supply 78, and 5 voltpower for the laser of reagent bar code reader 346 is provided by line562. 5 Volt DC power to the liquid crystal display 34 is provided byline 564. 24 Volt DC power is provided to the upper motor controller 566for the stepper motor 14 by line 568. 24 Volt DC power for the lowermotor controller 570 for the stepper motor 48 is provided from powersupply 76 by line 572.

The conventional card rack 529 has a separate 5 volt/12 volt powersupply 576. 5 Volt DC logic power and 12 volt DC motor power is providedto the floppy disc drive by lines 574.

FIG. 25 is a schematic drawing of a first portion of the computerdigital I/O system in the apparatus of this invention. The controlsystem uses a series of standard optical relays, each of which areconnected to close the line to ground in the power circuit for therespective component. The optical relays provide isolation.

Communication between the optical relays and the computer digital I/Oboard 580 is provided by lines 582. The two way solenoid valves 460, 462and 464 controlling the rinse liquid flow from heated rinse supply 44 tothe respective rinse blocks 200, 202 and 204 are energized to an openposition and de-energized to a closed position by output signals fromthe computer digital I/O board 580 to the optical relays 584, 586 and588. The two way solenoid valve 446 controlling the flow of evaporationcontrol liquid from container 434 to the nozzle block 212 is energizedto an open position or de-energized to a closed position by outputsignals from board 580 to optical relay 590.

The three way solenoid valves 412, 416 and 420 controlling air flow tothe respective tipper air cylinders 278, 280 and 282 are energized to anopen position (causing air flow) or de-energized to a closed position(venting cylinder air to the atmosphere)by output signals from computerI/O board 580 to respective optical relays 592, 594 and 596. The threeway solenoid valve 424 controlling air flow to the micro deliveryreagent dispenser control cylinder 300 is energized to an open position(causing air flow and reagent delivery) or de-energized to a closedposition (venting cylinder air to the atmosphere) by output signals fromcomputer I/O board 580 to respective optical relay 598. The two waysolenoid valve 468 controlling air flow to the vortex air mixer blocks220, 222 and 224 (FIG. 17) is energized to an open position (causing airflow to the mixer blocks) or de-energized to a closed position by outputsignals from computer I/O board 580 to respective optical relay 600.

The sound alarm 602 is activated to produce sound by an output signalfrom the computer I/O board 580 to optical relay 604. The sound alarm602 can be activated to sound a `beep` by keyboard key operation, by alonger `beep` or double `beep` at the completion of a run, and asustained sound during a system malfunction, for example. The three waysolenoid valve 432 controlling air flow to the rinse liquid andevaporation control liquid supply containers 44 and 434 (FIG. 22) isenergized to an open position (causing air flow and pressurization ofthe supply containers) or de-energized to a closed position (ventingcylinder air from the containers to the atmosphere) by output signalsfrom computer I/O board 580 to respective optical relay 606.

The slide heat fan 56 speed is operated by pulse width modulation, thatis, power pulses from the power relay 608. The fan 56 is energized by anoutput signal to the power relay 608 from optical relay 610. The timedsignal to the optical relay 610 is received from the computer I/O board580. The pulse width and speed of the fan 56 is adjusted in response toheating requests from the high temperature slide controller 632 toincrease the volume of heating air delivered to the air distributionmanifold 30.

The slide heater system control supplies separately controlled power toeach of the resistance heating elements 331 and 332. Low temperatureheating element 332 energized by power relay 612 upon a signal from thelow slide temperature controller 614. Thermistor 347 providestemperature information to the controller 614. During the operation ofthe apparatus at the lower temperatures required for theimmunohistological processes, the power to the heating element 332 isturned on when operating heat is required, in response to a lowtemperature signal from the low temperature controller 614. It is turnedoff when the operating temperature is restored. The controller 614 alsodetects when the slide door switch 616 is closed. If the cabinet slidedoor is open, energy supply to the heating element 331 and 332 isinterrupted. The heating cycle is initiated by a request for heat passedto the computer I/O board 580 through line 624 to the optical relay 622.The computer then responds with a heating power select heat signalreceived by controller 614 through line 620 from optical relay 618 inresponse to an output signal from the computer I/O board 580. A statussignal for the slide door switch is received by the computer I/O boardthrough line 628 and optical relay 626.

The high temperature heating element 331 is energized by power relay 630upon a signal from the high slide temperature controller 632, inresponse to a power command signal through optical relay 634 and line636 from the computer digital I/O board 580. During the operation of theapparatus at the lower temperatures required for the immunohistologicalprocesses, the power to the heating element 331 is turned on only duringan initial warm-up cycle. During the warm-up cycle, heat energy isrequested from the I/O board 580 through line 638 and optical relay 640.

When the apparatus is operated at the higher temperatures required forin situ hybridization, the heating elements are energized in a differentcontrol sequence by the controllers 614 and 632. As with the lowtemperature operation, both heating elements 331 and 332 are energizedduring the warm-up cycle. However, in the high temperature operatingmode, the low temperature heating element 332 is continuously energized,and energy is supplied intermittently to the heating element 331. In thehigh temperature mode, therefore, the optical relay 634 receives a powercommand signal from the I/O output board 580 when the high temperaturecontroller 632 signals that more heat is required. In addition to theheater controls described above, an additional thermostat is provided inthe heater circuit which turns the heater off if the heater temperaturereaches 160° C., for example if the fan 56 fails.

The rinse liquid heating system resistance heater 390 (FIG. 19) isenergized through power relay 642 upon a signal from rinse fluidcontroller 644. Thermistor 391 monitors the rinse fluid temperature, andthe controller 644 provides a signal indicating whether or not furtherheat energy is required. A heat request signal for heating liquid isreceived by the computer I/O board through line 646 and optical relay648. The computer responds with a heat select signal from the I/O board680 through relay 650 and line 652.

FIG. 26 is a schematic drawing of a second portion of the computerdigital I/O system in the apparatus of this invention. The computerdigital I/O board 580 receives a signal indicating closure of the airpressure switch 402 (FIG. 22) through line 670 and optical relay 672.The computer digital I/O board 580 receives a home signal from thereagent carousel metal proximity home sensor through line 676 andoptical relay 674 when the metal block 803 and the reagent carousel 10are in the home position. The computer digital I/O board 580 receives ahome signal from the slide support metal proximity home sensor 610through line 680 and optical relay 678 when the metal block 229 and theslide support carousel 24 are in the home position.

The reagent carousel stepper motor 14 is operated by reagent carouselstepper motor controller 690 in response to commands received from thecomputer digital I/O board 580. Command signals for steps (motoroperation) are received through line 692, and command signals for thedirection of operation are received through line 694. The stepper motorhas a high and low torque operating mode, the low torque mode beingeffected by switching a resistor into the control circuit. The hightorque mode is Used to move the motor through the number of stepsrequired to place a selected reagent bottle in the reagent deliverystation. The low torque mode is used as a brake to hold the reagentbottle carousel in a position. The low or high torque command signal isreceived by the reagent carousel stepper motor controller 690 throughline 698 and optical relay 696.

The slide support carousel stepper motor 48 is operated by slide supportcarousel stepper motor controller 700 in response to commands receivedfrom the computer digital I/O board 580 command signals for steps (motoroperation) are received through line 702, and command signals for thedirection of operation are received through line 704. This stepper motoralso has a high and low torque operating mode, activated in the same wayand having the same functions as the reagent carousel stepper motoroperating modes. The high torque mode is used to move the motor throughthe number of steps required to place a selected slide in a selectedtreatment zone. The low or high torque command signal is received by theslide support carousel stepper motor controller 700 through line 708 andoptical relay 706. When the door switch 616 shows an open door status,the step command signals to the stepper motors 14 and 48 are prevented.If the door switch 616 is opened during a biological processing run, anyincomplete stepper motor sequence is permitted to reach completionbefore further step command signals are blocked.

The keyboard 710 is a conventional pressure sensitive keyboard. Theswitches 720-726, 730-736, 740-746 and 750-756 are closed by manualpressure applied to the surface of an impermeable flexible plastic layerover the switches. The switches are isolated and protected under theplastic layer and are not fouled by moisture or debris from thelaboratory or operator.

In operation input lines 711, 712, 714 and 716 are each sequentiallyenergized for a brief period by the computer digital I/O board 580, andthe lines 718, 728, 738 and 740 are each sequentially polled during thisbrief period. If line 718 polls positive while line 716 is energized,closure of switch 720 is indicated. In a similar manner, closure ofswitch 722 is indicated by a positive poll of line 718 when line 714 isenergized, closure of switch 724 is indicated by a positive poll of line718 when line 712 is energized, closure of switch 726 is indicated by apositive poll of line 718 when line 711 is energized, and the like.

FIG. 27 is schematic drawing of the computer serial and floppy disk I/Osystem in the apparatus of this invention. The computer RS-232 I/O port770 sends polling signal to the slide barcode reader 231 and receivessignals indicating bar code information read through line 772.Similarly, the computer RS-232 I/O port 770 sends polling signal to thereagent carousel barcode reader 346 and receives signals indicatingbarcode information read through line 774. Signals to the liquid crystaldisplay 34 are sent through line 776 from the RS-232 I/O port 770. Thecomputer RS-232 I/O Port 770 receives an availability polling signalfrom the printer 550 and sends digital data to printer 550 through line778.

Immunohistological methods for which the apparatus of this invention areparticularly suitable are described in concurrently filed, commonlyassigned patent application Ser. No. 07/488,348, filed Mar. 2, 1990, theentire contents of which are hereby incorporated by reference. A typicalimmunohistological method, as carried out with the apparatus of thisinvention includes the following steps.

1) Preparing the slides, including applying a bar code to the slideindicating the immunohistological process to be used with the sample,and manually rinsing and applying evaporation inhibiting liquid to thetissue sample surface before placement in the apparatus to preventdehydration of the sample.

2) Inserting a batch of slides in the apparatus, mounting each slide ina slide support.

3) Closing the apparatus and beginning the treatment processing. Theapparatus heating system is in the warm-up mode until the heating airtemperature reaches the desired level.

4) A slide is rinsed in the first rinse station (FIGS. 11-14) in sevenrinse cycles. Each cycle includes applying a 500 μL pulse of rinseliquid followed by tipping the slide support to effect draining. Thissequence can be repeated for seven rinse cycles as the slide is moved toand pauses in each of the second and third rinse stations, for a totalof twenty-one rinse cycles, for example. The slide then is treated in aseven second stay in the evaporation inhibitor and reagent solutionapplication station (FIGS. 11, 14 and 15). An initial quantity of 500 μLof an evaporation inhibiting liquid such as dodecane is applied to theslide surface. Then 200 μL of reagent solution is applied to the slide.

As each slide poises in the reagent application zone, the appropriatereagent container is moved by the reagent carousel to the reagentapplication station, and a metered volume of reagent is applied to theslide. In being applied to the slide, the reagent liquid is applied tothe uppermost surface (the evaporation liquid layer). It then passesthrough the evaporation inhibiting liquid layer to the underlyingaqueous layer, a procedure which would not be possible with aconventional solid glass coverslip.

6) The slide is then passed to each of the vortex mixing stations (FIGS.11, 14, 16 and 17). Here vortex jets stir the reagent on the slidesurface under the file of evaporation inhibiting liquid. This procedurewould not be possible with a conventional solid glass coverslip.

7) The slide is then carried by the carousel, pausing as each slidesupport is sequenced through the same steps, until it returns to theinitial rinse station, where the cycle is repeated. The reaction betweenthe reagent and the tissue sample continues during this period, andslides in each of the following slide supports is subjected to the samesequence of rinse, application of evaporation inhibitor, application ofreagent, stirring, and incubation.

8) In a typical immunohistological process using a four phase processwith a peroxidase enzyme antibody label, a sequence total of fivedifferent reagents are applied as the tissue sample is passed five timesthrough the reagent application zone. In such a process, the firstreagent is a hydrogen peroxide solution required to eliminate endogenousperoxidase activity in the tissue sample. The second reagent is aprimary antibody which binds selectively with an specific epitope forwhich the sample is being tested. The third reagent is a biotin labeledsecondary antibody which binds preferentially with the primary antibodyremaining on the sample following the preceding incubation and rinsing.The fourth reagent is avidin labeled with an enzyme such as a peroxidaseenzyme, the avidin binding with the biotin label remaining on the samplefollowing the preceding incubation and rinsing. The fifth reagent is asubstrate solution which is converted by the peroxidase enzyme to form adetectable label such as a fluorophore or chromophore at the site of anyprimary antibody binding with the sample.

9) Following the conclusion of the substrate solution treatment andincubation, the slide typically is removed from the carousel,coverslipped with a glass coverslip and examined to determine the extentof primary antibody binding with the tissue sample.

FIG. 28 illustrates an alternative embodiment of the intermediatesection 4, including the slide support carousel 24 and the associatedslide treatment stations, which dispenses with the tipper rinse methoddescribed above and employs an alternative rinsing arrangement, usingstationary slide supports, as will be more fully described hereinafter.The carousel 24 is rotated, for example, in a clockwise manner, asindicated by the arrow shown in FIG. 28, so that each slide support 26Aand associated slide 234 is positioned in the rinse zone A, evaporatorinhibitor and reagent application zone B, and agitation zone C forsuccessive treatment and incubation as previously described above.

In the embodiment depicted by FIG. 28, an alterative embodiment of theslide support 26A is provided which does not pivot, but rather isfixedly supported in a predetermined position on the carousel 24 byscrews or the like and structured so that the associated slide 234 isheld substantially horizontally as best seen in FIGS. 29A-29B. Referringto FIGS. 29A-29B, the slide support 26A has a distal end 103A, which isjuxtaposed to the center of the carousel 24, and a proximal end 104,which is positioned adjacent to an outer circumference of the carousel24.

The support 26A comprises a support plate 102A having a raised terminalguide end platform 106, adjacent the proximal end 104A and a supportpost 107A, adjacent the distal end 103A. The platform 106A and the post107A cooperate to support the slide 234 in a substantially horizontalposition at a predetermined vertical distance with respect to raisedterminal guide tabs 108A and 109A between which the slide 234 ispositioned.

As best seen in FIG. 29B, the tabs 108A, 109A are provided with avertical length such that the upper surface of the slide 234 ispositioned above the upper ends of the guide tabs 108A, 109A while therespective lateral edges 111A, 113A of the tabs 108A, 109A engage thelateral sides of the slide 234, i.e., the tabs 108A and 109A do notextend a far as the upper surface of the slide 234 to preventwicking-off of any liquid on the upper surface of the slide 234 by thetabs 108A and 109A. The lateral edges 111A, 113A cooperate with the aguide edge 115A at the platform 106A to orient the slide 234 at apredetermined position with respect to the slide support 26A, and thusthe carousel 24, for treatment at the various treatment stations to bedescribe hereinafter.

A clamping arrangement, generally indicated at 118A, positioned at theproximal end 104A, clamps the slide 234 to the slide support 26A. Theclamping arrangement comprises a pair of supports 119A between which aslide engaging member 120A is pivotally supported. Spring 121A biasesthe slide engaging member 120A to firmly hold the slide 234 against theplatform 106A and post 107A. The slide support 26A permits easy loadingand unloading of the slide 234, firmly holds the slide 234 in place,does not interfere with the operation of the bar code reader andprevents or minimizes the wicking, i.e., surface tension, from drainingliquids off the slide 234.

An alternative embodiment of the rinsing arrangement forming the rinsezone A is employed in the embodiment depicted by FIG. 28 which replacesthe rinse blocks, and arrangement thereof, used with the tipper rinsemethod previously described with respect to FIG. 14. Referring to FIG.28, the rinse zone A employs a first rinse block 200A, having a singlewash block nozzle, as best seen in FIGS. 30A-30B, and a second rinseblock 202A, having a dual wash block nozzle, as best seen in FIG. 31.

The first wash block 200A is preferably positioned at the beginning ofthe rinse zone A and the second wash block 202A is preferably positionedat the end of the rinse zone A so that the first and second wash blocksare spaced from one another. The first wash block 200A pulses streams ofrinse liquid onto a slide upon entering the rinse zone A and due to themeniscus effect of the rinse liquid at the edges of the slide, builds upa layer of rinse liquid which remains on the slide. After apredetermined waiting period, set by the time it takes for the slidecarousel to transport a slide between the first and second wash blocks200A, 202A, the second wash block 202A uses pulsed streams of rinseliquid, alternately directed at one and then the other of thelongitudinal edges of the slides, to knock or sweep the previouslydeposited layer of rinse liquid off of the slide.

The rinsing arrangement depicted in FIG. 28 rinses or washes the uppersurface of the slides with streams or jets of pulsed rinsing liquid, forexample, water, so that a low volume of rinsing liquid is used toprovide a high degree of rinsing. Because the rinsing of the slides is akey limit to the sensitivity of the assays as background or noise isdirectly related to rinsing and sensitivity is the signal to noiseratio, the wash blocks 200A, 202A precede the application of the reagentand are a preferred feature of this embodiment of the invention.

Referring to FIG. 30A, the first wash block 200A comprises a dingle washblock nozzle 201A having a plurality of nozzle outlet openings 203A, forexample 10 or so openings, which each provide a pulsed stream of rinseliquid 204A which impacts the rinse liquid impact zone 236 of the slide234 as previously described. Due to the meniscus effect of the rinseliquid at the longitudinal edges 234P and lateral edge 234L of the slide234, a layer of rinse liquid 213A is built up on the slide 234 as aresult of the repeated pulsing of streams of rinse liquid during theoperation of the first wash block 200A.

As best seen in FIG. 30B, a nozzle axis 240A of the nozzles of block200A forms an angle b with the horizontal, this angle being between 15and 35 degrees, preferably substantially 25 degrees.

FIG. 31 illustrates the second wash block 202A which employs a dual washblock nozzle 205A comprising a lower set of nozzle outlet openings 206Aand an upper set of nozzle outlet openings 207A which respectivelydirect streams of pulsed rinse liquid towards one or the other of thelongitudinal edges 234P of the slide 234.

As with the first wash block 200A, the streams of pulsed rinsing liquid,from each of the lower and upper sets of nozzle outlet openings 206A and207A, preferably impact the slide 234 at the rinse liquid impact zone236 which is upstream on the slide 234 from the tissue sample (notshown) positioned thereon. This feature of the first and second washblocks 200A and 202A is important due to the fragile nature of thetissue sample positioned on the slide 234. By directing the streams ofpulsed rinsing liquid at the impact zone 236 of the slide 234, the rinseliquid is provided with laminar flow by the time the rinse liquidreaches the tissue sample. As a result, undue damage to the fragiletissue sample is prevented.

The upper set of nozzle outlet openings 207A is constructed so that theassociated streams of rinse liquid are off-set at an angle from thelongitudinal center line of the slide 234 so that the pulsed streams ofrinse liquid are directed toward one of the longitudinal edges 234P ofthe slide 234. The lower set of nozzle openings 206A is constructed sothat the associated streams of rinsing liquid are also off-set at anangle from the longitudinal center line of the slide 234 so that thepulsed streams of rinse liquid are directed toward the other one of thelongitudinal edges 234P of the slide 234. As a result of thisarrangement, pulsed streams of rinse liquid are alternately andrepeatedly directed to one and then the other of the longitudinal edges234P of the slide 234 as will be more fully described hereinafter.

Preferably, separate plumbing and valving are provided for each of thelower and upper sets of nozzle outlet openings 206A and 207A of the dualwash block nozzle 205A to permit independent operation thereof. Inoperation, wash block 202A directs streams of pulsed rinsing liquid, forexample from the lower set of nozzle openings 206A, toward a singlelongitudinal edge 234P of the slide 234 and after completion thendirects streams of pulsed rinse liquid, for example from the upper setof nozzle opening 207A, to the other longitudinal edge 234P of the slide234. This procedure is repeated and has the effect of sweeping orknocking the layer of rinse liquid 213A off of the slide 234.

As with the first wash block 200A, the nozzle axis 240 (not shown) ofeach of the upper and lower set of nozzle openings 207A, 206A forms anangle b (not shown) with the horizontal of between 15 and 35 degrees,preferably substantially 35 degrees for the upper set of openings 207Aand substantially 25 degrees for the lower set of openings 206A.

FIG. 32 illustrates an alternative embodiment of a vortex air mixer 220Awhich in this case is a single mixer. Each of the single vortex airmixers 220A is positioned at the inner radius of the slides 234 suchthat an gas jet or cone 356A of, for example, air or the like, blowsoutwardly adjacent one of the longitudinal lateral edges 234P of theassociated slide 234 to effect mixing in a manner similar to thatdescribed with respect to FIG. 17. More specifically, the gas. stream356A impacts the surface of the evaporation liquid surface layer 360 andmoves the underlying reagent solution in a circular path on the tissuesection.

Each vortex mixer 220A has a nozzle channel 350A, including a nozzleorifice 351A, which is supplied with pressurized air via a supplychannel 358A, the nozzle channel 350A preferably intersecting the supplychannel at a lower portion thereof. Pressurized air is supplied to thesupply channel 358A from a air supply conduit 352A (arrows indicatingthe flow of air to and from the mixer 220A) connected to a pressurizedair source (not shown). Each of the vortex mixers 220A can be suppliedwith pressurized air via a common supply conduit 352A which connects andsupplies each of the supply channels 358A of the plurality of mixers220A illustrated in FIG. 28.

As best seen in FIG. 28, there are, for example twelve, single vortexmixers 220A on the inner radius of the slides 234. The nozzle orifice351A of each of the mixers 220A is preferable positioned so that thecenter of the gas jet or cone 356A is approximately 2 mm above thesurface of the slide 234 and 4 mm in from the adjacent edge 234X of theslide. 234 as best seen in FIG. 32.

A first mixer 220A is preferably positioned at station S2 adjacent thereagent drop point station S1 and mixer 220A is positioned at stationS3, the mixers 220A at stations S2 and S3 directing the stream of air356A to opposite longitudinal edges 234P of an associated slide 234 sothat mixing is enhanced as described below.

The exact positioning of the remaining mixers 220A is not critical,these mixers 220A being positioned to provide a semi-continuous mixing.Additionally, each mixer 220A is spaced so that they alternate inblowing the right side and then the left side of the slide 234. That is,the even mixers blow up the right side of each slide 234 passing by andthe odd mixers blow up the left side or vice versa. This enhanceskinetic mixing, provides uniform coverage and averages out any possibletemperature differences across each of the slides 234. These featureslead to more rapid and reproducible staining than can be obtainedmanually.

Additionally, the intermediate section 4 of the embodiment of FIG. 28 isprovided with a bar code cleaner, generally indicated at 233A, forcleaning drops of liquid off of the bar codes 233 (FIG. 32) provided foreach of the slides 234 for identification purpose as previouslydescribed. It should be noted that the bar code cleaner 233A is equallyapplicable to the previously described embodiment of the inventionemploying the tipper rinse method described above. The bar code cleaner233A is positioned, for example, downstream from the reagent drop pointstation S1 just beyond the first vortex agitation zone C as best seen inFIG. 28 and upstream and adjacent to the bar code reader position (notshown).

The bar code cleaner 223A is illustrated in detail in FIGS. 33A-33B andcomprises a bar code nozzle 333A supplied with compressed air or thelike via a supply channel 334A which is connected to a compressed airsource (not shown) by supply conduit 335A. The bar code nozzle 333A issupported above the slide carousel 24 by support 336A, as best seen inFIG. 33B, and affixed to the stationary support plate 22 of theintermediate section 4. The nozzle 333A emits a stream or cone of air337A which blows across the bar code 233 of an adjacent slide 234attached to the associated slide support 26A. The stream of air 337Ablows drops of liquid off of the bar code 233 which otherwise interferewith the reading of the bar codes by the bar code reader.

As best seen in FIG. 33A, the nozzle axis 338A of the bar code nozzle333A forms an angle of about 45 degrees with the horizontal.Additionally, the stream of air 337A preferably strikes the bar code233A in the area of the side of the bar code 233A closest to nozzle333A.

Since the embodiment of the intermediate section 4 described withreference to FIG. 28 does not employ the tipper rinse method, any rinseliquid remaining on the slide after operation of the second wash block202A is drained from the upper surface of the slides 234 by a jet drain148A which is illustrated schematically by FIG. 34. The preferredposition of the jet drain 148A is at the last rinse station of the rinsezone A just prior to the reagent drop point station S1 as best seen inFIG. 28.

The jet drain 148A directs a fluid stream 150A of, for example air, atsubstantially a 45 degree angle to the longitudinal axis of anassociated slide 234 and across one corner of the distal end 104A of theassociated slide 234. The action of the fluid stream 150A acts to blow,aspirate or siphon the buffer remaining after the rinsing performed atthe rinse zone A as described above.

Except for the differences noted above the embodiment so described withrespect to FIG. 28 is the same as the apparatus described above inconnection with the tipper rinse method and is capable of operating andperforming the immunohistological methods as previously described.

We claim:
 1. A method for mixing a reagent solution layer having a firstarea, a second area and a center area on a tissue sample mounted on astationary planar support surface comprising stirring the reagentsolution by applying a first and a second gas stream to areas of thereagent solution layer between the center area of the reagent solutionlayer and an edge of the planar support surface, the first gas streambeing directed against the first reagent solution area and the secondgas stream being directed against the second reagent solution area, thefirst and second gas streams being in opposite directions, and the firstand second reagent solution areas being on opposite sides of the centerarea of the reagent solution layer.
 2. The method of claim 1 wherein thereagent solution layer is covered by a layer of an evaporationinhibiting liquid, the evaporation inhibiting liquid being substantiallywater-insoluble, substantially water-immiscible and substantiallynon-viscous; having a specific gravity less than water, and a boilingpoint above 100° C.; and being devoid of chemical characteristics whichwould significantly interfere with biochemical reactions carried out onthe sample.